- ORAL MICROBIOLOGY
Mochamad Fahlevi Rizal: ORCIDiD | Publons | ResearchGate | CrossRef | PubMed | Google Scholar | Scopus | WoS
Noboru Kaneko: ORCIDiD | Publons | ResearchGate | CrossRef | PubMed | Google Scholar | Scopus | WoS
Hiroshi Ogawa: ORCIDiD | Publons | ResearchGate | CrossRef | PubMed | Google Scholar | Scopus | WoS
Introduction Antibacterial ingredients with high safety and mild taste that can be used for mouthwash for children are required. Matcha is one of the natural beverages that is made from the leaves of green tea (Camellia sinensis) and widely consumed by Japanese and Asian people. This study aimed to evaluate the effectiveness of matcha in suppressing biofilm formation by mutans streptococci, typical cariogenic bacteria.
Methodology Five laboratory strains of Streptococcus mutans (serotype c, e and f) and Streptococcus sobrinus (serotype d and g) were used to evaluate the antibacterial effect of matcha. Matcha extract was added to bacterial cells in Heart Infusion broth supplemented with 1% sucrose (HIS). After incubation for 24 hours, the formed biofilm was dyed by Crystal Violet, and optical density at 490 nm was determined as the amount of biofilm formation. The effect of 0.02% chlorhexidine in HIS and purified water (MilliQ) in HIS were also measured as well.
Results The amounts of biofilm formation by all serotypes of mutans streptococci in matcha + HIS were significantly lower than those in MilliQ + HIS as were chlorhexidine in HIS, except S. sobrinus serotype g. The differences of amounts biofilm formation by all S. mutans (serotype c, e and f) and S. sobrinus serotype d in matcha + HIS and in chlorhexidine + HIS were not significant statistically.
Conclusion Matcha extract has an equivalent effectiveness of 0.02% chlorhexidine against most serotypes of mutans streptococci.
Keywords Streptococcus mutans, Streptococcus sobrinus, chlorhexidine, green tea, mouthwash.
Efforts to obtain antibacterial ingredients that meet the criteria of comfort, especially taste, are needed to improve the quality of the oral cavity. Oral health is inseparable from the normal flora balance of the oral cavity in order to prevent the occurrence of caries and gingivitis. The use of natural ingredients that are commonly consumed by the wider community in a place needs to be expanded in the functions of maintaining public health, especially oral health . The ease of getting it and the consumption habits of the community makes it easy for materials like this to be accepted by the community if proven to have good effects on oral health. Because caries and gingivitis are also a problem in children in many countries, especially with developmental disabilities, natural ingredients as an antibacterial for the oral cavity are urgently needed to be easily accepted by the child community, both in terms of safety and ease of use [2-4]. Each community has unique ingredients developed in its area, but has not been utilized optimally in addition to regular consumption of food and beverages .
Green tea (Camellia sinensis) is commercially available in the market in three forms: Loose leaf, bagged and powdered (matcha) . Matcha is generally consumed in Japan as a beverage that has high anti-oxidants such as flavonoids . Many natural foods and beverages that are commonly consumed by people in a region, such as honey in the tropical regions, olive oil, virgin coconut oil and others have been partially known to have antibacterial effects especially in the oral cavity . With many natural sources like ginger and garlic that also have flavonoid and are commonly used in Asia one needs to observe the antibacterial effect on oral bacteria . As a comparison, chlorhexidine has been used for many years as a strong antibacterial standard and is also used in dentistry for preventive measures and treatment but the taste becomes an issue especially for children .
The streptococci that are commonly related to the human caries process are Streptococcus mutans and Streptococcus sobrinus. These cariogenic bacteria are divided into 5 serotypes (c, e, f, d and g) . There has been no study on the antibacterial effects of matcha against each serotype of mutans streptococci. It is expected that the results of research related to antibacterial from natural sources can be used to prevent infection in order to minimize chemical substances . This study aimed to evaluate the effectiveness of matcha in suppressing biofilm formation by each serotype of mutans streptococci.
- Material and Methods
2.1. Preparation of antibacterial solutions
As a matcha solution, 5 gram of powdered green tea matcha (Itoen, Tokyo, Japan) was added to 100 mL of water at 100˚C and boiled for 15 minutes to extract. Prior to use in the laboratory, the solution was filtered using 0.20 µm Minisart filter (Sartorius, Göttingen, Germany). The chlorhexidine solution used in this study was 0.02% and was prepared from 20% chlorhexidine gluconate (Wako Pure Chemical, Osaka, Japan).
2.2. Culture of mutans streptococci
As mutans streptococci, we used 3 laboratory strains of S. mutans (serotype c, e, f) and 2 laboratory strain of S. sobrinus (serotype d, g) (Table 1). Each strain from -80 ˚C stock was cultured in Brain Heart Infusion (BHI) broth (Becton Dickinson Maryland, USA) at 37 ˚C overnight and then also sub-cultured in BHI broth. The bacterial solution was centrifuged and dissolved in Heart Infusion broth (Becton Dickinson Maryland, USA) supplemented with 1% sucrose (HIS) until it reached 1 x 106 CFU/ml which was then used for the biofilm assay as strain culture.
2.3. Biofilm assay
The biofilm formation of each strain was evaluated using a 96 well plate. Each plate was divided into 5 areas of 12 wells and labeled as MT for wells that were filled by matcha solution and strain culture, CHX for wells that were filled by 0.02% chlorhexidine and strain culture, MQ for wells that were filled by MilliQ and strain culture, NC for wells that were filled by milliQ alone as negative control and PC for wells that were filled by HIS and strain culture as positive control (Table 2).
After 24 hours of incubation, the culture supernatant was discarded, and each well was washed twice with 200 µL Phosphate Buffered Saline (PBS). A 200 µL volume of 0.5% crystal violet was then added and incubated at 37 ˚C for 15 minutes.
The remaining crystal violet solution was discarded, the biofilm was washed once with 200 mL PBS, and then 200 µL of 96% ethanol was added to each well. The optical density of the biofilm formation was then measured at 490 nm (OD490) using a microplate reader .
2.4. Statistical analysis
The OD490 values evaluated as biofilm formation were compared between NC, MQ and PC to confirm the validity of the measurement. Then OD490 values of MT, CHX, NC and MQ were compared to determine the differences in their effects on biofilm formation. In order to compare the differences, t-test and Bonferoni correction were used.
All statistical analyses were performed using SPSS statistics 19 (IBM, USA); p values of 0.05 or less were considered significant.
Fig. 1 shows OD490 values which mean amount of biofilm formation of NC (MilliQ alone), MQ (MilliQ + bacterial cells in HIS) and PC (bacterial cells in HIS) by S. mutans (serotype c, e and f) and S. sobrinus (serotype d and g), respectively. The OD490 of PC showed significantly higher values than MQ, and those of MQ showed significantly higher values than NC in all strains (serotype c, e, f, d and g).
Fig. 2 shows OD490 values which mean amount of biofilm formation of MT (matcha solution + bacterial cells in HIS), CHX (CHX solution + bacterial cells in HIS), NC (MilliQ alone) and MQ (MilliQ + bacterial cells in HIS), respectively. The OD490 of MT, CHX and NC showed significantly lower values than MQ and no significant difference was found between MT, CHX and NC in all S. mutans (serotype c, e and f) and S. sobrinus (serotype d). In S. sobrinus serotype g, the OD490 of MT showed lower value than MQ though the difference did not reach statistical significance. The OD490 of CHX and NC showed significantly lower values than MQ and MT in S. sobrinus serotype g.
The OD490 of PC showed significantly higher values than MQ and those of MQ showed significantly higher values than NC in all strains, which indicated that the biofilm assay used in this study could evaluate the amount of biofilm formation adequately. The OD490 of MT and CHX showed significantly lower values than MQ and the differences between MT, CHX and NC were not significant in all strains except S. sobrinus serotype g. This means that the antibacterial effect of matcha solution resemble CHX solution in most strains of mutans streptcocci.
This in-vitro research provides an overview of the antibacterial effects of matcha on various strains of mutans streptococci. Mutans streptococci are considered as typical cariogenic bacteria [14,15]. Mutans streptococci can be classified into five serotypes, with dominant serotypes being slightly different locally. The effect of matcha against S. sobrinus serotype g was not significant among all mutans streptococci strains of serotype examined in this study. However, in oral cavity of humans worldwide, S. sobrinus serotype g is not a type that found dominantly [16-19]. In other study S. sobrinus serotype g more resistance to chlorhexidine than S. mutans and S. sobrinus serotype d . Therefore, matcha is thought to have the effect of inhibiting biofilm formation for most of mutans streptococci in oral cavity. One of the cariogenic properties of these bacteria owes the high performance of glucan synthesis to sucrose, which contributes to the biofilm formation [21,22]. So, the effectiveness of matcha in suppressing the biofilm formation by mutans streptococci colonization was important to be evaluated in this study. The better antibacterial will suppress more types of strains, meanwhile caries are not only caused by a single bacterial but related to the biofilm activity involving many bacteria .
Chlorhexidine is a common ingredient as an antibacterial agent and is widely used for mouthwashes [24-28]. However, chlorhexidine has a taste issue, and it has problems such as side effects on the mucosa and tooth coloring, so it is not suitable for mouthwashes in children [29,30]. The concentration of chlorhexidine used clinically is 0.12% and 0.2%, while in this study using a concentration of 0.02%. It was proved that the concentration of 0.03% was as effective as 0.12% or 0.2% concentration clinically [31-33]. The result of this study revealed that the low concentration of 0.02% chlorhexidine can inhibit biofilm formation of mutans streptococci.
Matcha has long been used for drinking and the safety has been established. The requirements for mouthwash that can be used by children are related to safety issue [34,35]. It is also considered to have less problems with taste. Therefore, it is considered that matcha is suitable as an ingredient for mouthwash for children. The bacteria investigated this time are mutans streptococci and are strongly related to dental caries. However, there are many others bacteria in the oral cavity related to dental caries and other oral disease such as periodontitis. In this study, we did not consider the influence on other such cariogenic bacteria and periodontal disease related bacteria. Hereafter, it seemed necessary to investigate the antibacterial effect on those bacteria.
Matcha has the equivalent effectiveness of 0.02% chlorhexidine against most serotypes of mutans streptococci, namely S. mutans serotype c, e and f and S. sobrinus serotype d. It is suggested that matcha is a promising ingredient as a safe and effective for mouthwashes in children.
MFR: Idea, experimental design, data analysis, wrote the manuscript. NK: Performed biofilm and spectrophotometry assay, substantial contributed to writing manuscript. HO: Performed substantial contributed to writing manuscript.
The authors have no conflict of interest. This research funding by Division of Preventive Dentistry, Department of Oral Health Science, Graduate School of Medical and Dental Sciences, Niigata University. The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.
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